Cerebral ischeische-mic stroke was induced by mid-dle cerebral artery occlusion in four healthy beagle dogs using silicone plugs.. The cerebral cortex, basal ganglia, and thalamus wer
Trang 1Veterinary Science
†
The first and second author contributed equally to this work.
*Corresponding author
Tel: +82-2-450-4140; Fax: +82-2-444-4396
E-mail: parkhee@konkuk.ac.kr
Canine model of ischemic stroke with permanent middle cerebral artery occlusion: clinical and histopathological findings
Byeong-Teck Kang 1,† , Jong-Hwan Lee 2,† , Dong-In Jung 1 , Chul Park 3 , Su-Hyun Gu 1 , Hyo-Won Jeon 1 , Dong-Pyo Jang 5 , Chae-Young Lim 1 , Fu-Shi Quan 2 , Young-Bo Kim 5 , Zang-Hee Cho 5 , Eung-Je Woo 4 , Hee-Myung Park 1, *
1 Department of Veterinary Internal Medicine, and 2 Department of Anatomy, College of Veterinary Medicine, Konkuk
University, Seoul 143-701, Korea
3 Acupuncture & Meridian Science Research Center and 4 College of Electronics and Information, Kyunghee University, Yongin 446-701, Korea
5 Neuroscience Research Institute, Gachon University of Medicine and Science, Incheon 405-760, Korea
The aim of the present study was to assess the clinical
and histopathological findings in a canine model of
ische-mic stroke Cerebral ischeische-mic stroke was induced by
mid-dle cerebral artery occlusion in four healthy beagle dogs
using silicone plugs They showed neurological signs of
forebrain dysfunction such as reduced responsiveness,
head turning, circling, postural reaction deficits,
percep-tual deficits, and hemianopsia These signs gradually
re-gressed within 4 weeks without therapy On magnetic
res-onance imaging, T2 hyperintensity and T1 hypointensity
were found in the cerebral cortex and basal ganglia These
lesions were well-defined and sharply demarcated from
adjacent brain parenchyma with a homogenous
appea-rance No abnormalities of the cerebrospinal fluid were
observed At necropsy, atrophic and necrotic lesions were
observed in the cerebral cortex The cerebral cortex, basal
ganglia, and thalamus were partially unstained with
tri-phenyl-tetrazolium chloride Histopathologically, typical
features of infarction were identified in cortical and
thala-mic lesions This study demonstrates that our canine
mod-el resembles the conditions of real stroke patients.
Key words: dog, histopathology, ischemic stroke, MCAO, MRI
Introduction
Strokes have been reported rarely in dogs and cats
[1,6,7,11,13,16,21] They are likely underdiagnosed
be-cause of a lack of clinical suspicion, unavailability of
mag-netic resonance imaging (MRI) or computed tomography
(CT), and the wide array of presenting clinical signs [9] However, strokes are now being recognized with greater frequency in dogs due to the increased availability of MRI [13] Until recently, most reports of strokes in dogs have been limited to postmortem investigations of dogs that died
or were euthanized due to the severity of their brain infarct
or the suspected underlying cause for infarction [6] Thus, the true incidence and prognosis of strokes in dogs are unknown There is no proven preventive or treatment for stroke in dogs [10]
Even though suspected cases of stroke in dogs can now be studied antemortem, necropsy must be performed for de-finitive diagnosis It is also difficult to find effective diag-nostic or therapeutic methods for stroke in clinical patients during short periods because of presentation related to time
of onset, the limited number of cases, and restriction of continuous examination and monitoring after the improve-ment of neurological signs In human medicine, to over-come these difficulties, many animal models of stroke have been created using various techniques [23]; among these, some canine models have been developed [4,8,12,14, 15,18-20,24]
The aims of this study were to create a canine cerebral in-farction model by modifying previous methods [15,19] and to describe the clinical presentation, MRI features, prognosis, and histopathological findings of experimen-tally embolized dogs
Materials and Methods
Four healthy mature beagle dogs (all males, 3-4 years old, weighing 10 to 15 kg), which had been reared in a farm for
6 months after importation from a commercial laboratory animal company (Harlan Interfauna, UK), were studied Prior to arrival in our facility, all dogs were tested for
Trang 2ca-nine distemper virus by reverse transcription polymerase
chain reaction (RT-PCR), and for Toxoplasma and
Neospora by IgG antibody titer test, and were only
ac-cepted after testing seronegative They were screened for
metabolic diseases by complete blood count and serum
chemistry analysis, and for external and internal parasites
related to neurological diseases (ticks and Dirofilaria
im-mitis) After arrival, they were adapted and assessed daily
for neurological or behavioral abnormalities and general
health status for 2 weeks Each dog was housed in a single
cage and fed twice a day with commercial dry food at a
well-ventilated facility The surgical procedures and the
experimental protocol were approved by the Institutional
Animal Care and Use Committee (Konkuk University,
Korea) The approved study endpoint was 4 months
fol-lowing middle cerebral artery occlusion (MCAO) All
dogs were euthanized at this point Criteria for early
eutha-nization included: serious neurological or clinical
com-promise and inability of the animal to care for itself,
inabil-ity to self-feed after the initial recovery period, and
in-activity and lack of alertness for a continuous 24-h period
Animal preparation and monitoring
Dogs were restricted for 12 h prior to the induction of
anesthesia They were premedicated with atropine (Je-Il
Pharm, Korea) (0.02 mg/kg body weight, subcutaneously
[SC]) and acepromazine (Samu Median, Korea) (0.2
mg/kg body weight, intramuscularly [IM]), and were then
anesthetized 30 min later using propofol (Hana Pharm,
Korea) (5 mg/kg body weight, intravenously [IV]), orally
intubated, and mechanically ventilated Anesthesia was
maintained with isoflurane (Minrad, USA) at 2 to 3%
in-spired volume during surgery The tidal volume and
ven-tilatory frequency were adjusted to maintain a partial
pres-sure of arterial oxygen (PaO2) of 150 ± 50 mm Hg and a
partial pressure of arterial carbon dioxide (PaCO2) of 40 ±
5 mmHg Blood gases, glucose, and hematocrit were
meas-ured before, during, and after MCAO The fluid balance
was maintained by intravenous administration of 0.9%
so-dium chloride (Dai Han Pharm, Korea) Rectal
temper-ature was monitored continuously and maintained at
36-38oC throughout the surgery
Embolus preparation
The embolus was made as described previously [15,19],
with some modifications In brief, a silk suture (4-0)
(B.Braun Medical Industries, Malaysia) was passed into
the tip of a 20-gauge venous catheter (Becton Dickinson
Korea, Korea), lopped at the hub, and passed back out of
the tip Using a 3-ml syringe, silicone rubber (Dow
Corning, USA) with catalyst was injected into the hub of
the suture-containing catheter and cured for 24 h After
curing, the catheter was dissected from the
silicone-at-tached suture The embolus was made by cutting the cured
silicone to a length of 7 mm It was inserted into the tip of
an 18-gauge venous catheter; the suture was passed out of the hub and then coiled in a 25-ml syringe The syringe was connected to the embolus-containing catheter, and was then filled with 20 ml of physiological saline The plunger was then placed in the syringe
Surgical procedure
Cerebral ischemia was induced by MCAO as described previously [15,19], with some modifications Animals were positioned in right lateral recumbency Hair from the neck area that was to be surgically exposed was shaved, and the skin was thoroughly prepared with povidone-io-dine and alcohol scrub A strict, sterile surgical technique was utilized in all cases A cervical incision was made to expose carotid arteries By using blunt dissection and pal-pating the carotid pulse, the carotid sheath was exteriorized
at the level of bifurcation under the sternomastoideus muscle The common carotid artery was separated from the vagosympathetic trunk The internal and external carotid arteries were identified The common carotid artery was temporarily elevated using umbilical tape A 16-gauge ve-nous catheter was directly inserted into the left internal car-otid artery through the carcar-otid bulb The 18-gauge cathe-ter/25-ml syringe loaded with an embolus was inserted through the 16-gauge catheter The embolus was flushed into the internal carotid artery and up to the origin of the middle cerebral artery (MCA) by applying moderate force
to the syringe plunger The saline was injected at a total volume of 20 ml, at a rate of 2 ml per second Delivery of the embolus was confirmed by arterial back-flow in the syringe The traction on the common carotid artery was re-moved, and the catheters were also removed The neck in-cision was then sutured, exposing the remaining suture of the neck All dogs were permanently occluded until they were euthanized
Recovery
After surgery, the dogs were woken up, extubated, and then returned to the cages in the animal recovery room Butorphanol (Myungmoon Pharm, Korea) (0.4 mg/kg body weight, IM) and ampicillin (Unibiotech, Korea) (20 mg/kg body weight, IV) were administered to the dogs for
1 week to control pain and bacterial infection A floor heat-ing lamp was placed in front of each cage, and the radiant heat was directed to one side of the cage (not directly at the animal) Animals were observed continuously until they had fully recovered, for about 4 h in total The next day, they were transported to the holding area and periodically observed until euthanasia The incision was cleaned daily with chlorohexidine flush solution and bandaged for 1 week Diuretic therapy with mannitol (Daehan Pharm, Korea) (1 g/kg body weight, constant rate of IV infusion for 30 min) was also continued for the next 1-3 days
Trang 3Table 1 Physiological parameters
*Pre-MCAO
During MCAO
†
Post-MCAO PaO2 (mmHg)‡
PaCO2 (mmHg)§
Hematocrit (%) Glucose (mg/dl) Temperature (oC)
112.5 ± 18.4 38.3 ± 4.19 44.1 ± 3.2 97.5 ± 11.4 38.2 ± 0.78
115.3 ± 10.2 36.3 ± 5.32 46.6 ± 5.17 96.6 ± 10.2 38.1 ± 0.96
120.7 ± 17.2 40.6 ± 6.1 43.75 ± 6.29 91.2 ± 10.6 37.8 ± 0.69
*Examined at 1 h before the surgery; †examined at 1 h after the surgery ‡PaO 2 : partial pressure of arterial oxygen; §PaCO 2 : partial pressure of arterial carbon dioxide.
post-stroke as necessary
Neurobehavioral scoring
Neurobehavioral scoring was performed using a
stand-ardized categorical rating scale as described previously
[2,18]; scoring was performed for motor function (no
defi-cit = 1, hemiparetic but able to walk = 2, stands only with
assistance = 3, hemiplegic and unable to stand = 4,
coma-tose or dead, not testable = 4), consciousness (normal = 1,
mildly reduced = 2, severely reduced = 3, comatose or dead
= 4), head turning (absent = 0, posturing and turns toward
side of infarct = 1, unable to lift head, comatose, or dead =
1), circling (absent = 0, present = 1, does not walk or dead
= 1), and hemianopsia (absent = 0, present = 1, unable to
test because of reduced consciousness or death = 1)
According to this scoring, a completely normal dog would
have a total score of 2, and a dog with the most severe
defi-cits (comatose or dead) would have a total score of 11 Each
dog was assessed prior to anesthesia, daily during the first
week, and then weekly until euthanasia
MRI scanning
Scans were performed 2 days after occlusion All dogs
were fasted the night before the procedures as a precaution
for anesthesia They were pre-medicated and anesthetized
using the same procedure, described in animal preparation
and monitoring Anesthesia was adjusted to maintain
im-mobility during the scan The scanning was performed in a
3.0 Tesla research scanner instrument (Impedance Imaging
Research Center, Kyung-Hee University, Korea) The MR
coil was placed around the head, and the dog was placed in
the magnet for MRI to identify infracted regions T1- and
T2-weighted images (WI) were obtained in sagittal,
trans-versal, and dorsal planes
Imaging analysis
The ischemic lesion area was calculated from T2-WI
us-ing imagus-ing software (MRIcro, Version 1.40; Chris
Rorden, USA) For each slice, the higher intensity lesions
in T2-WI when the signal intensity was 1.25 times higher
than that of the counterpart in the contra-lateral brain lesion
were marked as the ischemic lesion areas The lesion
vol-ume was presented as a volvol-ume percentage of the lesion
compared with the contralateral hemisphere
Cerebrospinal fluid (CSF) analysis
CSF was directly obtained from each dog after MR
scanning It was collected by puncture with a 20-gauge
sterile, disposable spinal needle (Hakko, Japan) of the
cer-ebellomedullary cistern in lateral recumbency The
in-clusion criterion for the study was no iatrogenic blood
con-tamination during CSF collection A total of 2-3 ml of CSF
was collected into a plain, sterile tube without
anticoa-gulant It was used for routine diagnostic evaluation; the
erythrocyte and nucleated cell count was determined using
a standard hematocytometer chamber, cytocentrifuge cyto-logy, and estimation of the total protein concentration us-ing a urine dipstick (Young-Dong, Korea) These analyses were performed within 30 min after collection
2,3,5-triphenyl-tetrazolium chloride staining and histopathologic examination
Four months after surgery, all dogs were euthanized with sodium pentobarbital (Hanrim Pharm, Korea) (80 mg/ kg body weight, IV) The brains were carefully removed and dissected into coronal 2-mm sections The fresh brain sli-ces were immersed in a 2% solution of 2,3,5-triphenyl-tet-razolium chloride (TTC) in normal saline at 37oC for 30 min
Brain slices were placed in 10% paraformaldehyde in phosphate buffer After at least 72 h of immersion fixation, the slices dehydrated and embedded in paraffin Trans-verse sections (5 µm) were cut, stained with hematoxylin and eosin, and examined by light microscopy for histo-pathologic alterations associated with ischemic stroke
Results
Physiological parameters
In all dogs, anesthesia and physiological parameters were well maintained throughout the surgery Infection in or around the incision site and pain and discomfort were suc-cessfully treated with butorphanol There were no sig-nificant differences in the physiological parameters before, during, and after MCAO The mean and standard deviation
of each parameter are summarized in Table 1
Neurobehavioral deficits
All embolized dogs were slow to awaken No dogs met the criteria for early euthanization One dog (identification number (ID) 1) showed neurological signs of forebrain dysfunction, such as reduced responsiveness, head turning, and circling The other three dogs commonly showed other
Trang 4Table 2 Neurobehavioral scores of experimentally embolized dogs
Motor function
1 day
1 week
2 weeks
1 month
4 months
Consciousness
1 day
1 week
2 weeks
1 month
4 months
Head turning
1 day
1 week
2 weeks
1 month
4 months
1 1 1 1 1 2 2 2 1 1 1 0 0 0 0
2 2 1 1 1 2 2 2 1 1 1 1 0 0 0
2 1 1 1 1 1 1 1 1 1 1 1 0 0 0
2 2 1 1 1 2 1 1 1 1 1 1 0 0 0
Circling
1 day
1 week
2 weeks
1 month
4 months Hemianopsia
1 day
1 week
2 weeks
1 month
4 months Total
1 day
1 week
2 weeks
1 month
4 months
1 0 0 0 0 0 0 0 0 0 5 3 3 2 2
1 1 0 0 0 1 1 1 1 1 7 7 4 3 3
1 1 1 0 0 1 1 1 1 1 6 5 4 3 3
1 1 1 0 0 1 1 1 1 1 7 6 4 3 3
Fig 1 Transverse (A and B) and dorsal (C and D) T1-weighted and
T2-weighted MR images of the brain in an experimentally embolized dog (ID 2) Hypointense (A and C) and hyperintense (B and D) le-sions were found in lateral cortex (arrows) and caudate nucleus (arrow heads) In T2-weighted image, the well defined lesion was sharply demarcated from adjacent brain parenchyma with a homoge-nous appearance Swelling, midline shift, and suppressed left lateral ventricle and thalamus by mass effect were identified in all images
signs, such as walking into walls, postural reaction deficits,
perceptual deficits (menance response and facial
sensa-tion), and hemianopsia, as well as the abnormalities
ob-served in ID 1 These signs gradually improved within 4
weeks without therapy However, hemianopsia was not
re-solved prior to euthanasia The neurobehavioral scores of
each dog are summarized in Table 2
MRI findings
MRI scanning was performed in all dogs Increased
sig-nal intensity on T2-weighted images and decreased sigsig-nal
intensity on T1-weighted images were commonly found in
basal ganglia (all dogs), the left ventral cortex (ID 1), the
left ventrolateral cortex (ID 3 and 4), and almost the entire
left cortex of the cerebrum (ID 2) These lesions were
well-defined and sharply demarcated from adjacent brain
parenchyma with a homogenous appearance, and were
confined to gray matter However, occasional white matter
involvement was found in 3 dogs (ID 2-4) due to severe
gray matter changes Swelling and a midline shift of the
brain caused by mass effects were identified in the same
dogs (Fig 1) No other lesions were found in the thalamus,
brain stem, or cerebellum
Trang 5Fig 2 The ventral (A) and lateral (B) surface of the brain (ID 2)
4 months after MCAO by a silicone embolus exhibits remarkable
atrophy and necrosis (arrows) in the affected lateral cortex
Fig 3 Coronal section of the brain (ID 2) after TTC staining
demonstrate unstained lesion on thalamus (arrow) and atrophic changes (arrow heads) on the left lateral cortex
Fig 4 Microscopic features of the brain in an experimentally embolized dog (ID 2) (A) Thalamic lesion Necrotic neurons (arrows),
nuclear pyknosis, eosinophilia of the cytoplasm, and karyolysis were prominent H&E stain, ×400 (B) Cortex lesion Loss of tissue co-hesion, infiltration by leukocytes (especially polymorphonuclear leukocytes), congestion of small parenchymal blood vessels (arrow heads), and angioblastic proliferation were observed H&E stain, ×100
Imaging analysis
On the estimation of the percent lesion volume, ID 2 had
the largest volume (67.8%) The other three dogs had
sim-ilar lesion volumes at 16.9% (ID 1), 23.8% (ID 3), and 20%
(ID 4)
CSF analysis
In all of the samples obtained, no erythrocytes were
found and nucleated cells numbered less than 5 cells/µl On
cytological examination, mononuclear cells were
un-commonly observed without pleocytosis CSF protein
concentrations were found to be less than 30 mg/dl by
esti-mation with a urine dipstick
Necropsy, TTC staining, and histopathology
Atrophic and necrotic lesions were observed on the
ven-tral surface of the left cerebral cortex (ID 1) and the lateral
surface of the left cerebral cortex (ID 2-4) (Fig 2) In these
three dogs, lesions that were not stained with TTC were
commonly found in the basal ganglia, lateral cortex, and thalamus (Fig 3)
Microscopic examinations were performed in all dogs Hallmarks of infarction such as neuronal cell body shrink-age, pyknosis of nuclei, eosinophilia of the cytoplasm, and neuronal loss were observed in thalamic lesions of ex-perimental dogs (Fig 4A) In cortical lesions, loss of tissue cohesion, infiltration by leukocytes, neuronal loss, marked angioblastic proliferation, and congestion of small paren-chymal blood vessels were identified (Fig 4B)
Discussion
We have successfully implemented a canine model of per-manent embolic stroke Models of cerebral ischemia can
Trang 6be created in various animals There are advantages and
disadvantages of utilizing animals of different sizes to
study cerebral ischemic stroke [24] Small animals (e.g.,
mice, rats, gerbils) are often more cost-effective, and allow
for relatively simpler genetic modification and
mana-gement However, they have lissencephalic brains, and
thus may be quite different in terms of anatomy and
func-tional aspects compared to the human brain On the other
hand, large animals (e.g., cats, dogs, pigs, sheep, and
mon-keys) have gyrencephalic brains, which are structurally
and functionally similar to the human brain [23] Even
though primates would be the best model for human
ische-mic stroke among large animals, both ethical and econoische-mic
issues limit the widespread use of primates [18] Thus, we
selected the dog as an experimental animal for the study of
ischemic stroke because it is readily and economically
available, easy to care for, and have predictable
inter-current diseases
Experimental cerebral ischemia can be global,
hemi-spheric, multifocal, or focal The focal or regional cerebral
ischemic model was chosen because, in clinical practice,
those patients affected by an ischemic insult of this nature
potentially have the greatest capacity for recovery [4]
Until now, two major techniques have been used for the
production of experimental models of focal cerebral
ische-mia: 1) extravascular arterial occlusion by clips, ligatures,
or elecrocautery; and 2) intravascular occlusion of one
ma-jor cerebral blood vessel, such as the MCA, by the
in-tra-arterial injection of various embolic materials [12]
Because secondary problems such as transoperative
hypo-xia, meningitis, intra-temporal hematoma, subarachnoid
hemorrhage, intracranial infection, and hydrocephalus
re-sulted from mechanical manipulation and violation of the
dura, an extravascular arterial occlusion model requiring
intracranial surgery is likely to be suboptimal [18] MCAO
models do not require craniotomy, and have been used
ex-tensively because of their purported relevance to human
thromboembolic stroke [23] Even though the major
caus-es of canine infarction are not exactly known,
thromboem-bolic stroke may be typical in dogs [5] Thus, we created a
canine cerebral infarction model by MCAO and used a
sili-cone plug as an embolus
In previous studies [15,19], an embolus was inserted into
the internal carotid artery through an incision We injected
a silicone plug through a catheterized vessel instead of an
incision, and thus, the process of insertion and the control
of hemorrhaging could be easily performed without
suturing We also injected a larger volume (8 to 13 ml) of
saline than in previous studies [15,19] Using these
modi-fied procedures, we successfully lodged an embolus into
the MCA in all dogs Even though the number of dogs
ex-amined in our study was small, our model showed
con-tinuous reproducibility In previous studies, an embolus
was inserted into the MCA in 70% [15] and 78% [19] of
ex-amined dogs, respectively Because those studies used sev-eral dogs (55 [15] and 19 dogs [19]), results obtained from greater numbers of experimental dogs will be needed to verify the reproducibility of this model
In this study, significant differences of physiological pa-rameters were not found before, during, or after the surgery Some other studies using rats [25] and macaques [3] also showed no differences following occlusion Thus, these variables may not be closely related to canine ische-mic stroke However, other parameters, such as mean arte-rial blood pressure, cerebral blood flow, hemoglobin con-centration, and blood gases, should be studied in the future because they have not been well-studied and their relation-ship with stroke in canine patients or the experimental model is not yet known
MCAO can be maintained transiently or permanently ac-cording to the purpose of study Even though reperfusion was not allowed during the 4 months of the study, all dogs survived and improved over time Because the vertebral ar-teries of dogs assume a greater importance in terms of the total blood supply to the brain [5], it may be possible to pro-tect the brain against the effects of cerebral arterial occlu-sion during such long periods Thus, longer periods of oc-clusion may be needed to create sufficient ischemic lesions and allow for continuous reproducibility in transient and permanent occlusion canine models than in other animals Because the end of an inserted suture is exposed through the incision site in our model, reperfusion can be easily per-formed at any time without thrombolytic therapy; this is necessary in a thromboembolic model using autologous clots The optimal time of reperfusion in a transient canine model should be investigated through further study
In canine stroke, clinical signs are typically characterized
as peracute or acute at onset, focal, and nonprogressive [22] With forebrain lesions, the clinical signs may vary from simple disorientation to death A unilateral lesion will induce ipsilateral circling and head turning, hemi-in-attention syndrome, contralateral central blindness, con-tralateral ataxia, and proprioception deficits [17] Most dogs with ischemic stroke tend to recover within several weeks with only supportive care [5]
In this study, mental alteration, ipsilateral circling, and head turning were commonly observed in all dogs All signs were acute after awakening from anesthesia, and im-proved within 4 weeks Seizures were not observed in any
of the dogs In veterinary medicine, seizures have been re-ported, but are considered an uncommon manifestation of focal ischemic stroke in dogs [9] Three dogs (ID 2-4) with large lesions in the cerebral cortex showed contralateral proprioceptive deficits and permanent hemianopsia, as well as the commonly observed signs mentioned above To improve these signs, more time (1 to 3 weeks) was needed compared to the other dog (ID 1) Hemianopsia and motor dysfunction may not have been observed in ID 1 because it
Trang 7had a relatively small lesion on the occipital lobe related to
vision and the cerebrum Even though ID 3 had the second
largest lesion, the reduced level of consciousness that was
commonly shown in other dogs was not observed Based
on these findings, the prognosis of the canine model of
is-chemic stroke may be closely related to the initial severity
of the neurological deficits and the presence of secondary
pathological effects (extracellular edema and increased
in-tracranial pressure)
MRI is the most sensitive imaging modality for
diagnos-ing ischemic stroke [5] CT is also used in the diagnosis of
stroke, but is inferior to MRI in detecting ischemic
in-farction because of beam-hardening artifacts, inferior
con-trast display, and inability to provide detailed multiplanar
views [10] Because dogs have larger brains than other
common animal models, they are more amenable to study
with imaging modalities [18] The MRI findings in
ische-mic stroke result from the accumulation of water due to
cy-totoxic and vasogenic edema [10] Thus, the typical MRI
features of ischemic stroke include T2 hyperintensity and
T1 hypointensity, and these were well-observed in this
study Even though the thalamus is vulnerable to MCAO
and unstained lesions were clearly observed on TTC
stain-ing, no abnormalities were found on MRI MRI was only
performed at 2 days after the occlusion, and more time may
be needed to find thalamus lesions than the basal ganglia
and cerebral cortex The detection of ischemic tissue in the
peracute and early subacute stage is difficult; thus, other
techniques (e.g., diffusion-weighted imaging, perfusion-
weighted imaging, magnetic resonance angiography,
mag-netic resonance spectroscopy, and fluid-attenuated
in-version recovery imaging) can be applied [6,7,10]
Because the features of these techniques are not well
known in dogs, the canine model can be useful for imaging
as well as therapeutic study in veterinary medicine
CSF analysis is variable in dogs with ischemic stroke In
most cases, the CSF is either normal or reflects a mild
mon-onuclear or neutrophilic pleocytosis [16] No CSF
abnor-malities were observed in this study
MCA has tree-like branches that bring blood to the entire
lateral cortex of each hemisphere The central branches of
the MCA are the medial and lateral striata arteries The
striata supply the basal ganglia, internal capsule, and
thala-mus with blood [5] The areas of the brain that are
vulner-able to ischemic stroke include the hippocampus, cerebral
cortex, cerebellum, thalamus, and basal ganglia [16] In
this study, ischemic lesions could be identified in the
cere-bral cortex, thalamus, and basal ganglia using MRI,
nec-ropsy, and TTC staining These lesions were confirmed by
histopathologic examinations
In conclusion, we have developed a canine model of
is-chemic stroke that has a resemblance to real stroke
patients However, additional studies are needed to verify
the reproducibility and clinical time courses of this model
Until recently, most therapeutic (neuroprotective and neu-roregenerative), diagnostic, and preventive studies for is-chemic stroke have been performed using small animals If verified canine models are used in these fields, more effec-tive, promising, and reliable results can be obtained and ap-plied to human and veterinary medicine
Acknowledgments
This work was supported by the SRC/ERC Program (R11-2002-103) and a Grant (M103KV010026-07K2201- 02610) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology (MOST), Korea
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